Molecular Exclusion and Restricted Diffusion Processes in Molecular-Sieve Chromatography^*

Abstract

From experimental measurements of equilibrium solute partitioning and of corresponding column elution volumes with Sephadex gels, there appear to be two primary molecular sieving effects responsible for the commonly observed variation of elution position with molecular size. The first is a simple exclusion effect presumably due to dimensional heterogeneity of the gel interstices. This effect is of primary importance in the more tightly cross-linked G-75 and G-100 gels. The second effect is a steric and frictional interaction of the solute molecule with the gel matrix and plays an important role in the more loosely cross-linked G-200 gels and possibly also in the agar gels. For these columns a method of calibration is derived, involving one adjustable parameter, the effective gel pore radius. This parameter is calculated from the effluent peak position of a macromolecule of known Stokes radius. A column calibrated by this method can then be used for determination of Stokes radii or diffusion coefficients of other macromolecules and an upper limit for the hydrated molecular weight can be obtained. For systems that conform to the theory a separation coefficient for any two macromolecules of known radii or free diffusion coefficients can be calculated for a column of any effective gel pore radius. This separation coefficient then determines the column size necessary to obtain a desired separation volume between effluent peaks of the two molecular species.

Geometry of the Protein S4 from Escherichia coli Ribosomes

“…The Stokes' radius of protein S4 was determined from a logarithmic plot of Ro versus elution volume [erf-' (l-KD)], after calibration of the column with standard proteins of known Stokes' radius, by determining the distribution coefficient, KD, according to Ackers [25].…”

Tissue distribution and biochemical properties of an interspecific tumour-associated gamma foetal antigen

“…r: effective gel-pore radius (Ackers, 1964). cate analyses of y-FA from both em bryo and QUA tumour showed that the antigen from either source had about the same Stokes radius (Table II).…”

“…The peak of y-FA elution was determined by radial immunodiffusion assay of lOul aliquots from each fraction. The Stokes radius of y-FA derived from tumour and embryo was calculated according to Ackers (1964) using data obtained from duplicate analyses.…”

“…Average VO of 4 separate determinations was 16-4 ml. KD = VeV : distribution coefficient (Ackers, 1964).…”

“…42-2-16-4= 25-8 ml). a: Stokes radius; the known values for gamma-globulin, ovalbumin, myoglobin, cytochrome C, and bovine serum albumin were as reported (Ackers, 1964) or as calculated from the diffusion coefficient (Sherman, 1975).…”